Article pubs.acs.org/biochemistry
Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira bif lexa and Halorhodospira halophila L. Tyler Mix,† Julia Kirpich,† Masato Kumauchi,‡ Jie Ren,‡ Mikas Vengris,§ Wouter D. Hoff,‡ and Delmar S. Larsen*,† †
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, United States § Faculty of Physics, Laser Research Centre, Vilnius University, Sauletekio 10, LT-10233 Vilnius, Lithuania ‡
S Supporting Information *
ABSTRACT: We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PYP, but this residue is Ile in wild-type Lbif PYP. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PYP family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PYP photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PYP confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.
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The chromophore binding pocket of photoreceptor proteins often greatly increases the quantum yield and specificity for the alkene photoreceptor compared to those of the free chromophore in solution,10,11 while reducing fluorescence quantum yields. This issue is of primary importance for bioenergetics, biological light sensing, and the use of photoreceptors as light-activated switches in the emerging field of optogenetics.12 Optogenetics, using light to control biological processes, depends on having access to well-understood photoreceptors.13 We are studying the influence of the chromophore binding pocket on photoreceptor yield and dynamics using the photoactive yellow protein (PYP) as a model system. PYP is a small, 14 kDa, 125-residue, watersoluble protein with complex photochemistry that is initiated by the photoexcitation of an embedded p-coumaric acid (pCA) chromophore covalently attached to Cys69 (Scheme 1).14 In the dark-adapted pG state, the pCA is deprotonated with the double bond in its coumaryl tail in the trans configuration. Upon the absorption of a blue photon, an excited-state
unlight provides the energy necessary to support life in the Earth’s biosphere. Sunlight is responsible for driving photosynthesis processes and modulates a wide range of other important light-driven bioactivities, including phototaxis,1,2 gene expression,3 and vision.4,5 Moreover, photobiological processes are responsible for the mechanisms that modulate the expression of photosynthetic infrastructure via complementary chromatic adaptation.6 The transduction of light energy into usable function is performed by two distinct processes to accomplish transmembrane proton transfer in membrane proteins. The first process proceeds via electron transfer in chlorophyll-based photosynthetic reaction centers.7 The second process is driven by double-bond photoisomerization in the chromophore of retinal-based proteorhodopsins.8 The photochemical fate of the electronically excited state formed upon light absorption is different in these two classes of photoactive proteins. While the parameters that govern electron transfer are well understood,9 the factors that control chromophore photoisomerization to initiate the signaling process have been a topic of active interest. Proteins that use light energy to initiate biological signaling are termed photoreceptors, and this process is often initiated by chromophore photoisomerization. © XXXX American Chemical Society
Received: May 30, 2016 Revised: October 13, 2016
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DOI: 10.1021/acs.biochem.6b00547 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry Scheme 1. Local Protein Environment around the pCA Chromophore for Hhal PYP (left) and Lbif PYP (right)a
a
The residues shown in bold are different between the proteins, and the Ile68 residue (red) is modified to a Pro residue in the mutant Ile68Pro Lbif PYP.
isomerization reaction occurs that generates a cis conformation on a picosecond time scale.15,16 This triggers several groundstate steps, including an intramolecular proton transfer to the phenolic end of the chromophore and then large-scale structural changes in the protein through the alterations of hydrogen bonding interactions.17−19 The diversity of chemical reactions in PYP makes it a rich system for investigating how a protein structure and dynamics guide chromophore dynamics and vice versa. The vast majority of studies aimed at understanding the photoactivity of PYP have focused on the protein isolated from the proteobacterium Halorhodospira halophila (Hhal PYP),20 including mutants21−25 or Hhal PYPs expressed with modified chromophores.26 While close to 100 PYP homologues have been identified in genomes, few studies have been reported on the photochemical characterization of PYPs from these other organisms.27,28 Recently, we identified a PYP homologue in the spirochete Leptospira biflexa (Lbif PYP) with an amino acid sequence that is only 38% identical and 56% similar to that of Hhal PYP.27 The pCA chromophore pockets of Hhal PYP and Lbif PYP are qualitatively similar (Scheme 1) with three key differences. (i) Residue 68, which is immediately adjacent to Cys69, is an isoleucine in Lbif PYP but a proline in >90% of all PYPs identified to date.27 (ii) Residue 52 at the interface between pCA and the solvent is an arginine in Hhal PYP but a nonpolar valine in Lbif PYP. (iii) Residue 50 is a threonine in Hhal and an alanine in Lbif PYP. In Hhal PYP, the Thr50 residue contributes to the hydrogen bonding network between pCA and the surrounding protein scaffolding, while the Ala50 residue in Lbif PYP does not participate in the hydrogen bonding network. The hydrogen bonding network is integral in the formation of the lightadapted pB state.11 Substitutions at position 50 induces substantial red shifts in the absorbance spectrum of the protein in Hhal PYP,11,28,29 and this is presumably the origin of the redshifted spectrum in Lbif PYP (Figure 1). The role of Arg52 in the Hhal PYP photocycle is disputed as experiments with the Arg52Gln mutant showed a slight decrease in the speed of the reaction, but the mutant retained photoproduct production similar to that of WT Hhal PYP.22 However, molecular dynamics simulations by Groenhof et al. indicate that a positively charged Arg52 controls which of the bonds isomerizes in the pCA chromophore,30 while the mutant with Gln52 isomerized at the nearby single bond (C3−C1).31 Further experiments have argued that Arg52 in PYP is not
Figure 1. Steady-state normalized absorption spectra of Hhal PYP (black), Lbif PYP (red), and I68P mutant Lbif PYP (green) in the pG state with pCA in the trans conformation (Scheme 1). The laser spectrum of the 438 nm excitation pulses (gray) is indicated.
charged32 and may not influence the forward isomerization reaction for the Arg52Ala mutant.33 Because the photocycle proceeds with the Arg52Gln22 and Arg52Ala33 mutations, it is believed that the carbonyl flip and breaking the hydrogen bond with the amine backbone of Cys69 are the crucial actions in the initial photoreaction.17,31 A suggested role of Pro68 in WT Hhal PYP was that this residue constrict the chromophore pocket, leading to isomerization. When residues with smaller side chains are mutated into position 68,21,34 the photoreaction is slowed considerably. Groot et al. also reported that smaller residues have a more pronounced effect in decreasing the quantum yield in initiating the photocycle.21 While extensive studies have yielded a deep and detailed understanding of the molecular mechanisms of the initial steps of the PYP photocycle,15,17,35,36 important unresolved issues remain. First, the decay of the electronically excited state is triphasic, but the underlying quenching mechanisms are unclear. Second, the role of the residues surrounding the chromophore pocket and the influence of the apoprotein in non-Hhal PYP systems have not been explored. Third, Imamoto and co-workers reported evidence of cryogenic measurements for the existence of an early blue-shifted photocycle intermediate (at ∼450 nm), called PYPH.37,38 However, most other studies that reported ultrafast visible pump−probe experiments have found no evidence of such an early blue-shifted state.21−23,36 Here we report data that yield novel insights into all three issues, through ultrafast transient B
DOI: 10.1021/acs.biochem.6b00547 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry absorption on the first non-Hhal PYP protein, Lbif PYP, and through low-temperature kinetic different absorption experiments with Hhal PYP.
beam was modulated by a mechanical chopper to 500 Hz to collect difference spectra between the pumped and unpumped samples. The probe pulses were mechanically delayed from −10 ps to 7.5 ns with respect to the pump pulse by a computercontrolled linear motor stage (Newport IMS 600). The polarization of pump and probe pulses was set at 54.7° (magic angle) with respect to each other to eliminate anisotropic effects associated with rotational dynamics. The samples were passed continuously through a commercial quartz 1 mm flow cell (Starna) to provide fresh protein for each laser shot, and the sample was maintained at room temperature during all measurements. The resulting transient absorption (TA) signals had an ∼125 fs temporal resolution (instrumental response function), estimated by the rise of the excited-state absorption (ESA) band. The ultrafast broadband TA signals of Lbif PYP, I68P Lbif PYP, and Hhal PYP were measured consecutively on the same experimental setup and under the same experimental conditions to allow direct comparison of the data. The 438 nm excitation wavelength was selected to avoid multiphoton ionization of pCA.36,40 A cartoon of the experimental setup is available in Figure S1. Cryokinetic Spectroscopy. The low-temperature cryokinetic measurements were performed using an Oxford Instruments Optistat DN liquid nitrogen cryostat placed in the beam path of a Shimadzu UV−vis spectrometer. The PYP sample was dissolved in a solution of 66% glycerol and 33% buffer in a custom designed cell. The cryokinetic measurements were performed in two separate (back-to-back) experiments because of increased light scattering from the sample when the vitrified sample “cracks” at very low temperatures to generate a highscattering sample: (i) from 160 to 290 K and (ii) from 80 to 160 K. Experiments begin at the highest temperature, 290 or 160 K, and reference spectra are recorded every 3 min for 33 min. Next the temperature is “dropped” by 10 K, to 280 or 150 K. Each temperature drop is completed in
